Use of parasitic biological agents for prevention and control of autoimmune diseases

ABSTRACT

The invention relates to a method of treating an excessive immune response including an aberrant/enhanced Th1 response by administering a helminthic parasite preparation in an amount sufficient to reduce the excessive immune response in an individual. This invention is generally directed to autoimmune diseases which involve an excessive immune response or an aberrant/enhanced Th1 response. More specifically, the present invention is directed to the treatment of Crohn&#39;s disease and ulcerative colitis, both known as IBD. While the present invention discloses specific information about the treatment of IBD, the disclosure is in no way limiting. Additionally, rheumatoid arthritis, type 1 diabetes mellitus, lupus erythematosis, sarcoidosis and multiple sclerosis can be treated by the methods and compositions disclosed therein.

FIELD OF THE INVENTION

This invention relates to the prevention and/or treatment of a conditionor disease state in an individual which results from an excessive immuneresponse.

BACKGROUND OF THE INVENTION

Parasites are living entities that dwell on or in other creatures duringsome part of their life cycles, drawing nourishment from the host.Parasites that inhabit the intestines have a complex interplay with themucosal immune system. They must establish a tranquil relationship withhost mucosal defenses to survive. Helminths are elaborate multicellularworms with complex life cycles and development. The nematodes(non-segmented roundworms) and the platyhelminths (flatworns) are thetwo groups of helminths that colonize the human intestines. Perhaps morethan a third of the population of the world currently shelter one ormore of these organisms. The life-time exposure rate, however, isactually much more. The prevalence of helminths is highest in warmclimates and in populations subject to crowding, poor sanitation andimpure food supply. Inflammatory bowel disease (IBD), rheumatoidarthritis and autoimmune diseases are rare in these same regions.

Nematodes that frequently inhabit the human gut are Ascarislumbricoides, Enterobius vermicularis (pin worm), Trichuris trichiura(whipworm), Ancylostoma duodenale and Necator americanus (hookworms),and Strongyloides stercoralis. Trichinella spiralis infests the smallintestine briefly.

The platyhelminths include the trematodes and cestodes. The most commonadult trematodes that reside in the human intestines are Fasciolopsis,Echinostoma and Heterophyes species. Those that live in the biliarysystem include Clonorchis sinensis, Opisthorchis viverrini

The platyhelminths include the trematodes and cestodes. The most commonadult trematodes that reside in the human intestines are Fasciolopsis,Echinostoma and Heterophyes species. Those that live in the biliarysystem include Clonorchis sinensis, Opisthorchis viverrini and felineus,and Fasciola hepatica. Schistosoma dwell in the venous system, butseveral species chronically affect the gut by the passage of eggsthrough the intestinal wall. Adult cestodes commonly infecting humansare Diphyllobothrium species (fish tapeworm), Taenia saginata (beeftapeworm), Taenia solium (pork tapeworm) and Hymenolepsis nana (dwarftapeworm).

The host acquires various helminthic species through contact with soil,food or water contaminated with the infective form of the parasite.Children most frequently harbor helminthic infections because of theirclose contact with soil and suboptimal hygienic practices. Helminthsincite an intestinal Th2 response, which can cause worm expulsion orlimit the magnitude of infection. Most children living innon-industrialized countries have these parasites. Many helminthicspecies survive for years within the gut, biliary tree or mesentericveins making thousands of eggs daily. Thus, beginning in childhood,these worms and/or their ova release molecules that bathe the intestinalmucosal surface for years inciting Th2-type inflammation.

Dysregulation of the immune system leading to an excessive Th1 responsemay be the cause of several human diseases. Some diseases due todominant Th1 responses include IBD, rheumatoid arthritis, sarcoidosis,multiple sclerosis, and insulin-dependent diabetes melitis.

IBD is more common in temperate climates. It is not known what causesthe geographic differences. Observations suggest an environmentalexposure unique to temperate countries and highly industrializedsocieties predisposes to the development of IBD. An alternativeexplanation is that it is unhealthy to be raised in an “over clean”environment. It is proposed herein that the major environmental factorpredisposing to IBD is underexposure during childhood to intestinalhelminths, which promote strong Th2-type inflammation.

The frequency of CD has increased substantially over the last 40 years.It is most prevalent in temperate regions that are highlyindustrialized. This suggests that there is some critical environmentalfactor responsible for the change in frequency. Also, ulcerative colitisis rare in underdeveloped countries. It is proposed according to theinvention that the absence of exposure to intestinal helminthicinfections in childhood is an important environmental factor favoringthe development of CD and perhaps ulcerative colitis (UC).

People in industrialized countries are living in increasingly hygienicenvironments and are acquiring helminths much less frequently. Thedecreasing frequency of helminthic infections appears to correlated withthe increasing prevalence of CD. A case in point is the marked increasein the frequency of CD in young Asians and Africans after residing inIsrael for greater than 10 years. Also, the frequency of helminthicinfestation differs between the Jewish Israelis and Arabs. In 1969,stool examinations of hospitalized patients in Arab-predominant EastJerusalem contained helminthic ova over 60% of the time. The frequencyin Israeli-predominant East Jerusalem was 10% or less.

Thus, It is possible that the failure to acquire helminths and toexperience mucosal Th2 conditioning predisposes to CD and UC. There is aneed to combat CD and UC by re-colonization of the gastrointestinaltract with these organisms which may afford protection.

One object of the invention is to prevent or treat an excessive immuneresponse in an individual. The excessive or aberrant immune response canbe caused by an autoimmune disease, for example, IBD, rheumatoidarthritis, type 1 diabetes melitis, lupus erythematosis, sarcoidosis andmultiple sclerosis.

Further, it is an object of the instant invention to provide a method ofvaccinating an individual against autoimmune disease, for example, IBD,rheumatoid arthritis, type 1 diabetes melitis, lupus erythematosis,sarcoidosis and multiple sclerosis.

It is an object of the present invention to provide a method of creatingan immune environment in an individual that is conducive to amelioratingIBD.

It is an object of the present invention to provide a pharmaceuticalcomposition comprising a helminthic parasite and a pharmaceuticallyacceptable carrier.

It is a further object of the present invention to provide apathogen-free helminthic parasite and an acceptable pharmaceuticalcarrier.

It is another object of the present invention to provide a method ofpreparing pathogen-free helminthic parasites.

SUMMARY OF THE INVENTION

The instant invention presents a method of preventing or treating anexcessive immune response in an individual by administering an effectiveamount of an helminthic parasite preparation to reduce the excessiveimmune response in the individual.

The invention encompasses a method of treating an excessive immuneresponse, including an aberrant/enhanced Th1 response, comprisingadministering a helminthic parasite preparation in an amount sufficientto reduce the excessive immune response.

As used herein, the term “helminth parasite preparation” refers to anyone of a whole parasite, a parasite extract, parasite eggs, parasite eggextract, parasite larvae, parasite larvae extract, parasite cercariaeand parasite cercariae extract.

The helminthic preparation may be selected from the group consisting ofhelminths that naturally colonize humans and helminths that colonizeanimals but may protect humans from an excessive Th1 response.

In other preferred embodiments of the invention, the helminth parasiteis a nematode, and may be selected from the group such as Ascarislumbricoides, Enterobius vermicularis, Trichuris trichiura, Ancylostomaduodenale and Necator americanus, Strongyloides stercoralis andTrichinella spiralis.

In other preferred embodiments of the invention, the helminthic parasiteis a platyhelminth, and may be selected from the group consisting oftrematodes and cestodes, such as Fasciolopsis, Echinostoma andHeterophyes species, Clonorchis sinensis, Opisthorchis viverrini,Opisthorchis felineus, Fasciola hepatica, Schistosoma species,Diphyllobothrium species, Taenia saginata, Taenia solium andHymenolepsis nana.

In other preferred embodiments, the helminthic parasite is selected fromthe group consisting of filarial parasites and lung flukes.

In additional embodiments, the helminthic parasites are selected fromthe group consisting of Trichuris muris, Trichinella spiralis,Nippostrongylus prasiliensis, Heligrnonsomoides polygyrus, Hymenolepsisnanan, Angiostrongylus species, Trichuris suis, Ascaris suum, Trichurisvulpis, Toxocara species, Gnathostoma species, Ancylostoma species,Anisakis species and Pseudoterranova species.

The invention also encompasses a method of preventing or treating anautoimmune disease in an individual comprising administering ahelminthic parasite in an amount sufficient to prevent or treat theautoimmune disease in an individual.

As used herein, the term “autoimmune disease” refers to diseases such asIBD, rheumatoid arthritis, type 1 diabetes mellitus, lupuserythematosus, sarcoidosis and multiple sclerosis

The “effective dosage” amount of the helminthic parasite preparation isdependent upon the specific cause for the excessive or aberrant immuneresponse, and is addressed in detail below.

As used herein, the term “excessive” or “aberrant” immune responserefers to a T helper cell type 1 (Th1) response in which the activity ofT helper 1 cells is elevated in an individual relative to the activityof such cells in an individual who is not affected by the disease.Typically, the elevation of the Th1 response in the diseased individualwill be at least 2-fold, and possible 5-fold-10-fold above the Th1response in an individual who is not diseased. Th1-type inflammationsproduce large amounts of IFN-γ and TNFα, which in turn stimulate astrong cellular immune reaction. These are some of the cytokines thatmay be measured to indicate an excessive or aberrant Th1 response, asdescribed in detail hereinbelow.

Preferably, the helminthic parasite is selected from the groupconsisting of parasite, parasite extract, parasite eggs, parasite eggextract, parasite larvae, parasite larvae extract, cercariae andcercariae extract.

The invention also encompasses a method of treating IBD comprisingadministering a helminthic parasite preparation in an amount sufficientto reduce IBD.

The invention also encompasses a method of vaccinating an individualagainst a disease involving an excessive immune response comprisingadministering a helminthic parasite preparation in an amount sufficientto prevent the excessive immune response.

The invention also encompasses a pharmaceutical composition comprising ahelminthic parasite preparation, and a pharmaceutically acceptablecarrier.

Further features and advantages of the invention will become more fullyapparent in the following description of the embodiments and drawingsthereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The figure describes the concentrations of IFNγ, IL-4 and IL-5measured in spleen cell supernatants of mice infected with M. avium, S.mansoni or both organisms. Splenocytes (4×10⁵/well) were cultured invitro for 48 h at 37° C. in 200 μl of medium in the presence or absenceof optimal concentrations of PPD or SEA. Cytokine secretion wasquantified by ELISA.

FIG. 2. The figure describes the concentrations of IFNγ and IL-4 ingranuloma cell supernatants of mice infected with M. avium, S. mansonior both organisms. Granuloma cells (4×10⁵/well) in 200 μl of medium werecultured in vitro at 37° C. for 48 h in the presence or absence of theoptimal concentration of PPD or SEA. Cytokines were quantified by ELISA.

FIG. 3. The figure describes the serum IgG1, IgE and IgG2a levelsmeasured in mice infected with M. avium, S. mansoni or both(concurrent). Immunoglobulin concentration was determined by ELISA.

FIG. 4. The figure shows that IL-4 treatment can cure mice of a chronicH. polygyrus infection. Mice received three injections of an IL-4complex starting 12 days before killing. Data are means +/−SE ofmultiple determinations.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the recognition that diseases thatinvolve excessive or aberrant Th1 immune responses are treatable byadministration of a helminthic parasite preparation.

The invention relates to a method of treating an excessive immuneresponse including an aberrant/enhanced Th1 response by administering ahelminthic parasite preparation in an amount sufficient to reduce theexcessive immune response in an individual. This invention is generallydirected to autoimmune diseases which involve an excessive immuneresponse or an aberrant/enhanced Th1 response. More specifically, thepresent invention is directed to the treatment of Crohn's disease andulcerative colitis, both known as IBD. While the present inventiondiscloses specific information about the treatment of IBD, thedisclosure is in no way limiting. Additionally, rheumatoid arthritis,type 1 diabetes mellitus, lupus erythematosis, sarcoidosis and multiplesclerosis can be treated by the methods and compositions disclosedtherein.

The entire disclosure of each of the literature references citedhereinafter are incorporated by reference herein.

Helminthic Parasites Useful According to the Invention

In defining a helminthic parasite, there are two groups. The first groupis helminthic parasites that naturally colonize humans and the secondgroup is helminthic parasites that colonize animals, but may affordprotection to humans.

In the first group, helminthic parasites are elaborate multicellularworms with complex life cycles and development. The nematodes(non-segmented round worms), and the platyhelminths (flat worms) are twogroups of helminths that colonize the human intestines. In accordancewith the present invention, any one of a number of helminth parasitesthat naturally colonize humans or animals will provide the intendedresults.

Nematodes that frequently inhabit the human gut are Ascarislumbricoides, Enterobius vermicularis (pin worm), Trichuris trichiura(whipworm), Ancylostoma duodenale and Necator americanus (hookworms),and Strongyloides stercoralis. Trichinella spiralis infests the smallintestine briefly.

The platyhelminths include the trematodes and cestodes. The most commonadult trematodes that reside in the human intestines are Fasciolopsis,Echinostoma and Heterophyes species. Those that live in the biliarysystem include Clonorchis sinensis, Opisthorchis viverrini and felineus,and Fasciola hepatica. Schistosoma dwell in the venous system, butseveral species chronically affect the gut by the passage of eggsthrough the intestinal wall. Adult cestodes commonly infecting humansare Diphyllobothrium species (fish tapeworm), Taenia saginata (beeftapeworm), Taenia solium (pork tapeworm) and Hymenolepsis nana (dwarftapeworm).

Other helminths of interest include the filarial parasites and the lungflukes. These do not have a gut phase, but stimulate strong Th2-typeresponses.

The second general group of helminthic parasites that can be utilized inthe present invention include helminths that colonize animals, but mayafford humans protection against Th1-mediated diseases. These includeTrichuris Muris (mouse whipworm), Trichinella spiralis, Nippostrongylusbrasiliensis, Heligmonsomoides polygyrus and Hymenolepsis nana, all ofwhich are intestinal helminths that infect mice. Additionally,Angiostrongylus is a rat helminth. Trichuris suis and Ascaris suum arepig helminths that can infect humans. Trichuris vulpis, Toxocaraspecies, Gnathostoma, and Ancylostoma are dog or cat helminths that alsocan infect humans. Anisakis and Pseudoterranova are nematodes of marinemammals that can transmit to humans. Bird schistosomes can transientlyinfect humans. Such schistosomes include S. douthitti, Trichobilharziaocellata, T. stagnicolae, T. physellae, and Gigantobilharzia huronensis.

Diseases Treatable According to the Invention

A. Inflammatory Bowel Diseases (CD and UC):

Epidemiological data suggest a genetic susceptibility to the developmentof Crohn's disease (CD) and ulcerative colitis (UC). The incidence of CDin industrialized societies has increased from the 1950s until the mid1980s, and now is from 1 to 8 per 100,000 persons per year. Thissuggests that unknown changes in our environment have effected thefrequency of CD.

While the cause of IBD remains undetermined, it is presumed to resultfrom dysregulation of the intestinal mucosal immune system. Inflammatorycells in the mucosa normally protect us from luminal contents. Thishighly effective chronic inflammation is tightly controlled to limittissue injury. IBD may result from inappropriately vigorous immuneresponses to luminal factors. CD appears to be an overly vigorousTh1-type inflammation that produces IFN-γ and TNFα. The nature of UC isless well defined.

There are several animal models of chronic intestinal inflammation, forexample, as reported by Elliott et al. (Elliott et al., 1998,Inflammatory Bowel Disease and Celiac Disease. In: The AutoimmuneDiseases, Third ed., N. R. Rose and I. R. MacKay, eds. Academic Press,San Diego, Calif.). An important advance is the recent discovery thatsome mice with genetically engineered gene deletions can develop chronicbowel inflammation similar to IBD, see Elson et al., 1995,Gastroenterology 109:1344. These include mutant mice bearing targeteddeletions for IL-2, IL-10, MHC class II or TCR genes among others. Usingsome of the models, Berg et al., 1996, J. of Clin. Investigation98:1010, Ludviksson et al., 1997, J. of Immunol. 158:104, and Mombaertset al., 1993, Cell 75:274 have shown that a dysregulated immune systemitself can mediate intestinal injury. The mucosal inflammation ofseveral of these models generates large amounts of IFN-γ and TNF-αsuggesting that excess production of Th1-type cytokines is one commonmechanism underlying the pathogenesis of disease. Also, blocking Th1circuitry prevents the inflammation. CD is a Th1 response. Thus, thesemodels may have direct implications regarding the immunopathology ofthis human disease process.

B. Rheumatoid Arthritis (RA):

RA is a chronic disease featuring persistent inflammatory synovitis,usually involving peripheral joints in a symmetric distribution. Thisinflammation can lead to bone erosions, cartilage damage and jointdestruction. It is an affliction of about 1% of the population. Theprevalence increases with age, and women are affected more frequentlythan men. The propagation of RA is an immunologically mediated eventdriven by CD4+ Th1 cells.

C. Insulin-Dependent, Juvenile Diabetes Mellitus (DM) (Type 1):

Type 1 DM is a disease that usually begins during early adulthood andthat results from the inability to produce insulin in response toincreasing blood sugar. This persistent high blood sugar and inabilityto properly metabolize glucose causes metabolic disturbances thateventually damage the eyes, kidneys, heart and other organs. Takinginsulin parenterally can partially control these metabolic problems.Type 1 DM results from an autoimmune attack on the pancreatic betacells, which are the source of insulin. Activated macrophages andcytotoxic T cells surround and destroy the pancreatic beta cells.Genetic susceptibility and poorly defined environmental events triggerthe disease process.

D. Lupus Erythematosus (LE):

LE is a systemic autoimmune disease that is most frequent in women ofearly to middle adulthood. The tissue damage is caused by autoantibodiesand hyper-reactive regulatory T cells. The abnormal immune responseallows sustained production of pathogenic autoantibodies and immunecomplexes. This leads to damage of the musculoskeletal, cutaneous,hematologic, renal and other systems. The abnormal immune responseprobably depends upon the interaction of multiple hereditary andenvironmental factors.

E. Sarcoidosis:

Sarcoidosis is a chronic granulomatous disease of the lungs and otherorgans of unknown cause. Most patients present between the ages of20-40. The most frequent symptom is shortness of breath. The diseaseresults from an exaggerated Th1-type, cellular immune response, probablyto a limited number of antigens. Sarcoidosis develops throughout theworld, and afflicts all races. However, there is remarkable diversity ofthe prevalence of sarcoidosis among certain ethnic and racial groups.For instance, the disease is rare in Poland, Southeast Asia and India.

F. Multiple Sclerosis (MS):

MS is a chronic relapsing, multifocal inflammatory disorder of thecentral nervous system that leads to focal demyelination and scarring ofthe brain. It is a frequent disease affecting about 350,000 Americansand which begins during early to middle adulthood. MS is an autoimmunedisease mediated at least in part by Th1 cells. The lesions of MSresemble those induced by delayed hypersensitivity responses thatcontain activated T cells and macrophages. It is a disease of temperateclimates, increasing in prevalence with distance from the equator.

Helminthic Parasite Preparations According to the Invention

A. Viable Organism Vaccines:

Viable helminthic parasite organisms may provide the most profound Th2mucosal conditioning because of their relative longevity as compared tocomponent vaccines. Viable organisms can be administered in either egg,larval, cercarial, or encysted larval forms depending on the helminth.Helminths that can colonize humans and a preparatory animal may beutilized.

The preparatory animal may need manipulation to allow high patency bythe helminthic. Such manipulation can include treatment with agents thatare immunosuppressive like glucocorticoids or azathioprine; agents thatimpede Th2 effects like anti-histamines, anti-cytokines, or recombinantcytokines; and agents that influence intestinal motility likeanti-cholinergics or opiates. The animal's diet will be altered toreduce coarse fiber content. The animal may be a rat, pig, hamster, birdor other preparatory animal.

Preparatory animals are raised in specific pathogen-free (SPF)environments according to methods known in the art. They are tested toensure absence of human bacterial, mycobacterial, and viral pathogens.

Eggs: Some intestinal helminths are acquired by ingestion of viableeggs. Helminths are maintained in SPF preparatory animals, for example,SPF pigs. To harvest eggs, the animals are given a special diet low incoarse fiber. Animals are given an oral purgative to induce defecation.Stool is collected and enzymatically digested to free eggs. Eggs areisolated from liquefied stool by flotation on density gradients, screenfiltration, Visser filtration, or centrifugal elutriation. Preservationof eggs varies with the helminth used. Eggs from helminths that areresistant to dessication are dried, compounded with inert material,incorporated into an enteric capsule, and refrigerated. Eggs fromhelminths that are susceptible to dessication are preserved byrefrigeration in liquid medium or by adding cryoprotectant and freezingin liquid nitrogen. Viable eggs are washed, mixed with chilledlactose-free pudding or other vehicle at the location for delivery. Eggsstored in glycerol-based cryoprotectant may not require washing. Eggsfrom each lot are tested for hatching rate to determine effectivedosing. Eggs from each lot are tested for absence of bacterial and viralpathogens.

Larvae: Some helminths (i.e. hookworms) require a soil maturation phasebefore they can colonize humans. Eggs from these agents will beincubated under optimal conditions to mature the embryo, or hatch theegg and provide larval forms. Patients can be inoculated by subcutaneousinjection of viable larvae.

Cercariae: Some helminths have complex life cycles that utilize anintermediate host. The intermediate host sheds the form able to colonizehumans. Cercariae are the form for trematode helminths (i.e. flukes)shed by intermediate hosts like snails. Cercariae are isolated fromcolonized snails grown in SPF conditions. Cercariae are washed. Thesemay be preserved by adding cryoprotectant and freezing in liquidnitrogen. Thawed or fresh cercariae are washed and injectedsubcutaneously to inoculate patients. Samples from each lot are testedfor absence of pathogens and to determine effective dose.

Encysted Larvae: Some helminths (e.g. tapeworms) form encysted larvae orcysticerci in intermediate hosts. It is the encysted larval form thatinitiates human colonization. Encysted larva are removed fromintermediate hosts, for example, cattle or fish or plants grown in SPFconditions. Cysts are washed free of remaining host tissue. Cysts may bepreserved by adding cryoprotectant and freezing in liquid nitrogen.Cysts are thawed or used fresh, washed, mixed with chilled lactose-freepudding or other vehicle at the location for delivery and fed toindividuals. Samples from each lot are tested for absence of pathogensand to determine effective dose.

B. Non-Viable Component Vaccines:

Non-viable components of helminthic parasites provide sufficient Th2conditioning of the immune response to prevent Th1-mediated pathology.Non-viable components are derived from eggs, larvae or adult worms.

Non-viable, intact schistosome eggs produce a strong Th2 response. Eggsare isolated from livers of preparatory animals (i.e. hamsters) grownunder SPF conditions. Eggs are isolated by a method modified from thatoriginally described by Coker and Lichtenberg, 1956, Proceedings of theSoc. For Exp. Biol. & Med. 92:780. The modifications consist of usingphosphate buffered saline with glucose instead of 1.7% saline forincubation and washing steps along with decreasing the autolyticdigestion time. These changes promote isolation of viable eggs. Themethod is as follows.

Infect golden hamsters with 1000 to 1500 cercariae. Allow the infectionto mature (6 to 7 weeks). Remove the livers from the animals and placein 600 mOsm sterile phosphate buffered saline containing 5% glucose, 100U/ml penicillin and 100 mg/ml streptomycin. The livers are allowed toautodigest for 24 hours at room temperature. Pulse homogenize the liversat low speed for 3 minutes in a cold Waring blender. Incubate thehomogenate with collagenase (2 mg/ml) and trypsin (2 mg/ml) at 32° C.for one hour. Filter the homogenate through 50 and 80-100 mesh sieves toremove clumps of tissue and debris. Recover the eggs from the filtrateby passing over a 325 mesh sieve. The eggs will not pass through thescreen. Flush the eggs off of the screen and into a 50 ml polypropylenecentrifuge tube. Wash the eggs by repeated low speed (400×g)centrifugation in sterile phosphate buffered saline with 5% glucose. Theeggs must be free of any collagenous debris. Count an aliquot of eggs ina 1 ml Sedwick chamber to determine total egg number.

Isolated eggs are suspended in saline and flash frozen in liquidnitrogen without cryoprotectant. This kills the egg. Thawed eggs areinjected subcutaneously, intramuscularly or intravenously, or at sitesof Th1 inflammation to elicit strong Th2 responses. Eggs from otherhelminths may also be utilized.

Component vaccines may also be used that employ proteins, lipids, orcarbohydrates isolated from parasite eggs. An example is schistosomesoluble egg antigens (SEA). The method for preparing schistosome eggantigen has been previously described by Boros and Warren, 1970, J. ofExperimental Med. 132:488 and is briefly given below.

Washed eggs are resuspended at 50,000 eggs/ml of phosphate bufferedsaline. This is transferred to a glass tissue homogenizer. The eggs arehomogenized on ice. To insure that all shells are broken and miracidiaare disrupted, an aliquot (5 ml) is removed for microscopic inspection.Transfer the homogenate to ultracentrifuge tubes. Centrifuge at100,000×g for 2 hours at 4° C. Recover the aqueous fraction (SEA) anddetermine the protein content. Store the SEA in small aliquots at −70°C. This method may require modification to isolate the parasite eggproducts that most strongly promote Th2 conditioning, i.e., to achievean optimal effective concentration, (100 μg SEA or 10,000 ova/animal).Eggs or soluble egg components are used to initiate Th2 responses or toboost Th2 responses previously initiated by colonization with viablehelminths. Eggs or egg components are tested to confirm the absence ofpathogens and endotoxin.

Component vaccines can also be developed from larvae and adult worms ofhelminthic parasites. Larvae or worms are isolated from preparatoryanimals grown in SPF conditions. Vaccines that employ non-viable intactorganisms or proteins, lipids, or carbohydrates isolated from thehelminth are prepared and utilized in a manner similar to thatpreviously described for helminth eggs.

C. Maintenance of Helminth Organisms:

Helminths are cycled through intermediate and preparatory animals grownin SPF conditions. Samples of helminth populations are tested to ensurephenotypic stability such as colonization rates, fecundity, andsusceptibility to anti-helminthics.

Dosage, Administration and Pharmaceutical Formulations

Depending upon the helminthic parasites selected, treatment is deliveredby one of several approaches. Individuals in need of treatment receivethe infected form of the parasite (egg, cercariae or larvae) orally orparenterally depending upon the natural life cycle of the parasiteselected. Alternatively, soluble worm or egg extracts can be givenorally or parenterally to induce TH2 responses.

With regard to intestinal and liver helminths and schistosomes, theybegin producing ova that appear in the stool about 30-60 days afterinoculation. Quantifying the eggs in the stool proves satisfactory forassessing adequacy and intensity of infection. Aliquots of stool areprocessed by sucrose floatation to determine the total number of eggs ineach specimen. Flotation over sucrose solution is a method frequentlyused to isolate eggs from stool for accurate counting as reported byKoontz and Weinstock, 1996, Gastroenterology Clinics of N. America25:435.

The helminthic parasite compounds of the invention may be formulated foradministration in any convenient way, and the invention thereforeincludes within its scope pharmaceutical compositions comprising thehelminthic parasite compound in accordance with the invention adaptedfor use in humans. Such compositions may be presented for use inconventional manner with the aid of any necessary pharmaceuticalcarriers or excipients.

The helminthic parasite compound according to the invention may beformulated for injection, and therefore, vaccine use, and may bepresented in unit dose form in ampules, or multidose containers, ifnecessary, with an added preservative. The compositions may also takesuch forms as suspensions, solutions or emulsions of oily or aqueousvehicles and may contain formulatory agents such as suspending,stabilizing, and/or dispersing agents. Alternatively, the helminthicparasite may be in powder form or reconstituted with a suitable vehicle,e.g. sterile-pyrogen-free water, before use.

If desired, such powder formulation may contain an appropriate non-toxicbase in order to ensure that the powder is reconstituted with water, thepH of the resulting aqueous formulation being physiologicallyacceptable.

The helminthic parasite compounds may also be formulated assuppositories, e.g. containing conventional suppository bases such ascocoa butter or other glycerides.

The helminthic parasite compounds may also be formulated to oral dosagewith conventional fillers, carriers, and excipients. The amount ofparasite administered to the individual in need thereof is an amountsufficient to prevent or treat the autoimmune disease. This amount mayvary depending upon the disease being treated or prevented and thehelminthic parasite, whether it is being administered intact, or as anegg, larvae, extract or cercariae.

Typically, when the parasites are administered for all autoimmunediseases discussed herein, the amount ranges from about 50 to about50,000. More particularly, this amount may range from about 500 to about5,000. When eggs are utilized, about 500 to about 5000 may be utilizedto treat the autoimmune diseases disclosed herein. When extracts areadministered, about 100 μg to about 10,000 μg are utilized to treat theautoimmune diseases. When larvae and cercariae are administered, thedosages may range from about 500 to about 5,000 in each case.

For prevention or vaccine use, the amounts of the parasites may be500-5,000.

D. Determination of Th1 and Th2 Responses

In order to show the efficacy of the present invention, the Th1 and Th2response must be distinguished. Metawali et al., 1996, J. of Immunol.157:4546 has shown that in mice, it is possible to distinguish a Th1from a Th2 response by histologic analysis, and by analysis of cytokineand immunoglobulin profiles. Further, Sandor et al., 1990, J. of Exp.Med. 171:2171 has shown that cell surface expression of Fcg3 and MHCClass II molecules afford discrimination. In this procedure, small boweland colon are examined histologically to determine the degree of mucosalinflammation, eosinophilia and mastocytosis. The latter cell types areindicative of a Th2 response. Mesenteric lymph nodes (MLN) and spleenscan be dissociated into single cell suspensions for in vitro culture inmicrowell plates. Cells (1−2×10⁷/well) in complete RPMI medium arecultured for up to 72 h in the presence or absence of worm antigen oranti-CD3 and then the supernatants are assayed for cytokines andimmunoglobulins. IFN-γ, TNFα and IgG2a characterize a Th1 response,whereas IL-4, IL-5, IgE and IgG1 typify a Th2 reaction. Also, serum canbe assayed for cytokine and immunoglobulin concentrations. Furthermore,dispersed inflammatory leukocytes are examined by flow cytometry forFcγ3 expression on macrophages (Th1) and MHC Class II expression on Bcells (Th2). Controls include serum, MLN and spleens from appropriateage-matched, littermate mice that hosted no parasite. Also, there areother markers of the Th1 vs Th2 responses.

A similar analysis can differentiate a human Th1 from a Th2 response.One examines inflamed tissue, isolated leukocytes from regions ofinflammation and peripheral blood cells. Leukocytes are cultured invitro alone or in the presence of parasite antigen or mitogens tostimulate cytokine release. IgG2 substitutes for IgG2a.

Cytokine Detection by Flow Cytometry: Splenocytes, MLN or intestinalinflammatory cells in RPMI complete medium are placed into 24-welltissue culture plates at 2×10⁶ cells/well. Cells are incubated 4-6 h inthe presence or absence of anti-CD3 or appropriate antigen withbrefeldin A at 10 μg/ml. Brefeldin prevents exocytosis of proteins andpromotes accumulation of the cytokine within the cell. For cytoplasmiccytokine detection, the cells are fixed in 2% paraformaldehyde at roomtemperature for 5 min following surface staining to distinguish cellsubtypes. Cells are washed and re-suspended in 50 μl PBS 0.2% Saponinand 1 μg anti-cytokine antibody and incubated at room temperature for 20minutes. Next, the cells are washed twice in Saponin and re-suspended inPBS/FCS. The specificity of the cytokine antibody staining is confirmedby pre-blocking the cells with an excess of un-conjugated antibody ofthe same isotype and cytokine specificity or by incubating the cells inthe presence of recombinant cytokine. Phycoerythrin (PE)-labeledirrelevant antibody controls also are included to assess backgroundstaining. The cells are analyzed using flow cytometry.

ELISAs: ELISAs measure cytokine and antibody concentrations in cellsupernatants from cells cultured in microtiter plates and manipulated asdescribe above. Many of these assays are already operational within ourlaboratory. Cytokines are assayed using two monoclonal antibodies (mAbs)in a two-site sandwich ELISA. The anti-cytokine mAbs are purified byammonium precipitation from supernatants of antibody secreting hybridomaclones. Microtiter plates are coated with 50 μl of 1 ug/ml coatingantibody in PBS containing Tween 20 (PBS-T), and incubated at 4° C.overnight. Then, wells are blocked by the addition of 150 μl of 10% FCSin PBS with incubation at 37° C. for 30 min. Standards compriserecombinant cytokine or cytokine-containing supernatants from ConA-activated spleen cells from schistosomiasis-infected mice. Sample andstandard dilutions are made in RPMI containing 10% FCS (complete RPMI)in a separate 96-well flat bottom microtiter plate, and 50 μl volumesare transferred to the ELISA plates that have been washed three times inPBS-T. Samples are incubated in the assay plates for 1 h at 37° C.Appropriate mAb is conjugated to biotin. After washing three times inPBS-T, each well will receive 50 μl of antibody-biotin conjugate at 0.5μg/ml in 1% BSA/PBS-T. Plates are incubated at room temperature for 1 hfollowed by washing three times in PBS-T. Streptavidin-horseradishperoxidase conjugate (75 μl) is added at 1 μg/ml in 1% BSA/PBS-T andincubated at room temperature for 1 h. Plates are washed 10 times infresh PBS-T, and 100 μl of substrate (2.2′-azino(3-ethylbenzthiazolinesulfonic acid) at 1 mg/ml in 44 mM Na₂HPO₄, 28 mM citric acid, and0.003% H₂O₂ is added. The colored product is measured at a wavelength of405 nm with a reference wavelength of 490 nm, using a multiscanmicroplate reader.

Immunoglobulins are quantified using anti-isotype specific ELISAs.Affinity-purified goat anti-IgM, -IgG1, -IgG2a, -IgG2b, -IgG3, -IgA and-IgE are used as capture antibodies and absorbed to flexible polyvinylmicrotiter dishes at 10 μg/ml. After addition of culture supernatants,incubation and washing, appropriate isotypealkaline-phosphatase-conjugated goat anti-Immunoglobulin is used todetect total mouse Immunoglobulin bound to the plates. Standard curvesare generated using purified Immunoglobulin. To measure parasiteantigen-specific antibody, soluble antigen is biotinylated and used todetect bound mouse Immunoglobulin. The plates are analyzed on a ELISAreader at 410 nm, and concentrations of total Immunoglobulin aredetermined using the standard curve and best-fit analysis software.Antigen-specific antibody concentrations are compared relative to theO.D. readings, since soluble parasite antigen is not a defined antigenthat permits precise quantitation.

ELISPOT Assays: ELISPOT assays are established to count lymphocytessecreting either polyclonal antibody or cytokines. Ninety-six-wellnitrocellulose-backed microtiter plates are coated overnight at 4° C.with 1 g/ml of either anti-Immunoglobulin or anti-cytokine antibodies inPBS-T. The plates then are blocked with PBS containing 10% FCS andwashed extensively with PBS-T.

Serial dilutions of a single cell suspension, starting with 5×10⁴cells/well, are incubated on the plate for 5 h at 37° C. in a humidified5% CO₂ atmosphere. The plates are washed with PBS-T and overlaid withbiotinylated anti-Immunoglobulin or -cytokine antibodies overnight at 4°C. Next, plates are washed and treated with streptavidin-glucoseoxidase-conjugate for 2 h and washed again.

The antibody or cytokine secreted by single cells is visualized withsubstrate. The calorimetric reaction is halted after 30 min by washingand spots enumerated under 30× magnification. The dilution of cellsproducing 10-50 spots/well is used to calculate the total number ofsecreting cells per sample. Controls include wells coated withinappropriate goat antibody or inappropriate antigen, or left uncoated.

A modification of the assay using soluble antigen to coat the wellspermits quantitation of parasite antigen-specific, Immunoglobulinsecreting B cells also. Briefly, for example, plates are coated withadult T. Muris antigen at 0.25 μg/well or appropriate irrelevant controlantigen. Cells are added to the wells after washing. After appropriateincubation, the plates are washed again and treated as described above.

Statistical Methods Some data are analyzed by the one sample t-test todetermine whether mean values significantly differ from zero. Pairedt-test is used to analyze differences between group means. Analysis ofvariance and Dunnett's t-test is used to analyze multiple comparisondata.

Evidence of the presence of an autoimmune disease and its cure oramelioration is needed to determine the need for treatment and tomonitor treatment progress. The following procedures are utilized tomeasure the clinical parameters of the noted diseases.

1. Inflammatory Bowel Disease

Evaluation of Inflammation: In mice, clinical evidence of diseaseincludes weight loss, diarrhea, rectal prolapse and histologicalevidence of intestinal inflammation. Thus, improvement in theseparameters would signify amelioration of disease.

To grade intestinal inflammation in animal models, tissue is removed,Swiss-rolled and embedded in paraffin according to standard methods. Thesections are stained with hematoxylin and eosin. The degree of colonicinflammation is graded semiquantitatively from 0 to 4 in a blindedfashion by a single pathologist using our usual standardized technique:0=no inflammation; 1=low level inflammation; 2=intermediate levelinflammation; 3=high level inflammation with wall thickening; and4=transmural infiltration, and loss of goblet cells with wallthickening.

To count mast cells, intestinal tissue samples from individual mice areprepared by the Swiss-roll technique, fixed in Camoy's fixative,paraffin embedded and processed for staining with Alcian Blue andsafranin. Fifty adjacent fields of a given section are scanned formucosal mast cells in the lamina propria and muscle layers. Mast cellsare identified by their distinctive intracellular granular staining withAlcian Blue. All samples are evaluated blindly.

Disease activity in humans is monitored using various clinical,laboratory and histological criteria. There are several well establishedIBD disease activity indices that monitor clinical parameters likefrequency of diarrhea and abdominal pain. One particularly useful indexfor the assessment of Crohn's disease is the Crohn's Disease ActivityIndex, or CDAI (Best et al., 1976, Gastroenterology 70: 439). The CDAIincorporates 8 variables related to the disease activity and has beenused in most recent studies of therapeutic agents in Crohn's disease. Itincludes the number of liquid or very soft stools, the severity ofabdominal pain or cramping, general well-being, the presence ofextraintestinal manifestations of the disease, presence or absence of anabdominal mass, use of antidiarrheal drugs, hematocrit, and body weight.The composite score ranges from 0 to about 600. Scores below 150indicate remission and scores above 450 indicate severe illness.

A tested, accepted and disease specific quality of life questionnairealso may be administered prior to and after treatment to assesstherapeutic progress. The Irvine Inflammatory Bowel DiseaseQuestionnaire is a 32-item questionnaire. It evaluates quality of lifewith respect to bowel function (e.g. loose stools and abdominal pain),systemic symptoms (fatigue and altered sleep pattern), social function(work attendance and the need to cancel social events) and emotionalstatus (angry, depressed, or irritable). The score ranges from 32 to224, with higher scores indicating a better quality of life. Patients inremission usually score between 170 and 190.

Also, helpful are endoscopic, x-ray and histological assessment ofintestinal disease activity. C-reactive protein levels and blood cellsedimentation rate may also be monitored as systemic indicators ofinflammation.

2. Rheumatoid Arthritis

Evaluation of Inflammation:

For mice with collagen-induced arthritis, mice are examined every otherday and their paws scored as follows: 0, normal; 1, Erythema and mildswelling confined to the ankle joint or toes; 2. Erythema and mildswelling extending from the ankle to the midfoot; 3, Erythema and severeswelling extending from the ankle to the metatarsal joints; and 4,Ankylosing deformation with joint swelling. These four parameters can becorrelated with the histological changes in the arthritic joints.Treatment success results in a decrease in the arthritis score withimprovement in the histology.

For pristane-induced arthritis, joints may be measured with a micrometerto detect swelling. In humans, RA is scored by measuring joint swelling,erythema, limitation of motion and pain. Additionally, synovial fluidmay be analyzed for cytokine and inflammatory protein concentrations,and for leukocyte composition and function, according to methods knownin the art. Synovial biopsies provide tissue for histological analysisaccording to methods known in the art.

3. Lupus

Evaluation of Inflammation:

The normal development and function of the immune system criticallydepends on the removal of unwanted cells by a process called apoptosis.Cell-to-cell interactions through specific cell surface molecules andtheir receptors frequently trigger the process. One such system iscalled FAS and FAS ligand. Mice deficient in either FAS (LPR−/−) or FASligand (GLD−/−) develop an autoimmune disease like lupus.

Colonies of LPR or GLD mice are maintained in micro-isolator housingunits under specific pathogen-free conditions. These mice can developautoimmunity spontaneously, but more predictably after artificialinduction. To induce disease, 8-wk-old mice are injected with an agentlike pristane. Within two months, the mice have autoimmune disease. Manyclinical, histological and immunological criteria useful for judgingdisease induction and amelioration in both mice and humans are wellknown in the art.

4. Juvenile Insulin-Dependent Diabetes Mellitus (Type 1)

Evaluation of Inflammation:

The NOD mouse develops type 1 diabetes mellitus similar to humans due toautoimmune destruction of the pancreatic β cells. Clinical, biochemical,immunological and histological examination according to methods known inthe art allow assessment of disease induction and amelioration in mice.

5. Sarcoidosis

Evaluation of Inflammation:

In the bead embolization model of pulmonary inflammation, antigens (i.e.Th1 or Th2) are coupled to Sepharose beads, which are embolized to thelungs of mice via injection into their tail veins. The animals usuallyare pre-sensitized to the coupled antigen. The immune system of the hostmounts a vigorous immune response to the offending bead. These focalinflammatory responses, which can last for several weeks, can beexamined histologically for size. Also, they can be isolated from tissueand studied for cell composition and cytokine production. Moreover,hilar lymph nodes and spleens are readily available for experimentation.

Sarcoidosis, a disease of humans, usually involves the lung.Determination of sarcoidosis and the extent of the disease may be madeaccording to methods known in the art. Pulmonary function tests canassess lung compliance and function. Also, bronchiolar lavage obtainsinflammatory cells that have infiltrated into the bronchial tree duringthe inflammatory process. These cells can be studied for composition andfunction. Pulmonary infiltrates and hilar lymphadenopathy arecharacteristic of sarcoidosis. Thus, periodic chest x-ray or CT scanscan help assess disease activity. Serologic tests, such as measurementof angiotensin converting enzyme activity according to methods known inthe art, can be used to gauge disease extent and activity.

6. Multiple Sclerosis

Evaluation of Inflammation:

Experimental autoimmune encephalomyelitis is induced in susceptible miceby repeated injection of appropriate sensitizing myelin antigens. Miceare assessed clinically according to the following criteria: 0, nodisease; 1, tail atony; 2, hind-limb weakness; 3, hind-limb paralysis;4, hind-limb paralysis and fore-limb paralysis or weakness; 5, moribund.For histological analysis, the spinal cords and brains are removed andfixed in formalin. The paraffin-embedded sections are stained andexamined under light microscopy. Dispersed splenocytes and cells fromother regions can be studied in-vitro as outlined above. Theseparameters can help measure disease amelioration or improvement.

In humans, MS disease activity is gauged by monitoring progression andremittence of neurological signs and symptoms. The most widely usedoutcomes measurement is called The Expanded Disability Status Scale.Cerebral spinal fluid protein composition and cell content analyzedaccording to methods known in the art also may be used to monitordisease activity. Moreover, serial MRI studies show newgadolinium-enhanced brain lesions.

Animal Models Useful According to the Invention

The following Table I illustrates the disease conditions that can beprevented or treated by the helminthic parasites in accordance with thepresent invention. TABLE 1 TREATABLE DISEASES SOME ANIMAL MODELS 1.Inflammatory Bowel Diseases TNBS colitis (Neurath et al., 1995, J. ofExp. Med. 182: 1281) IL-2 mutant mice (Ludviksson et al., 1997, J. ofImmunol. 158: 104) IL-10 mutant mice (Berg et al., 1996, J. of Clin.Investigation 98: 1010) TCR transgenic mice (Mombaerts et al., 1993,Cell 75: 274) CD45+ T cells transferred into SCID mice (Powrie et al.,1994, Immunity 1: 553) 2. Rheumatoid Arthritis Murine pristane-inducedarthritis (Stasluk et al., 1997, Immunol. 90: 81) Murinecollagen-induced arthritis (Horsfall et al., 1997, J. of Immunol. 159:5687) 3. Insulin-dependent Diabetes (type 1) NOD mouse (Cameron et al.,1997, J. of Immunol. 159: 4686) 4. Lupus (NZWX NZB) F₁ models (Santiagoet al., 1997, J. of Exp. Med. 185: 65) GRD, LPR mouse (FAS mutation)(Bhandoola et al., 1994, Int. Rev. of Immunol. 11: 231) 5. SarcoidosisMurine Berylliosis (Pfeifer et al., 1994 Int. Archives of Allergy &Immunol. 104: 332) M. avium mouse (Chen et al., 1994, Science 265: 1237)6. Multiple Sclerosis Experimental allergic encephalomyelitis in mice

Mechanism of Action

This invention involves the prevention and/or treatment of autoimmunediseases in individuals by the administration of helminthic parasites,and to the prevention or vaccination and/or treatment of an excessiveimmune response or aberrant immune response in an individual due to IBD,Crohn's disease (CD), ulcerative colitis (UC), rheumatoid arthritis(RA), insulin-dependent, juvenile diabetes mellitus (DM) (type 1), lupuserythematosus (LE), sarcoidosis and multiple sclerosis (MS) byadministration of helminthic parasite to that individual.

Without being bound to any one mechanism of action, the followingmechanism is proposed.

Inflammation is a complex process involving many cell types and therelease of various immunoregulatory molecules called cytokines. Thetypes of cytokines secreted determine the nature of the inflammatoryresponse. Immune responses usually present as either Th1 and Th2.Th1-type inflammations produce large amounts of IFN-γ and TNFα, whichstimulate a strong cellular immune reaction to invading pathogens likebacteria, viruses and protozoa. A Th2-type response contains IL-4, IL-5,IgE, IgG1, mast cells and eosinophils. This type of inflammation is seencommonly in allergic reactions. Strong Th1 and Th2 responses aremutually exclusive. IFN-γ prevents expression of IL-4, and IL-4 impedesIFN-γ production, which are the two cytokines centrally important forthe Th1 and Th2 patterns of inflammation. Also there are other cytokineslike IL-10 and TGF-β that inhibit IFN-γ synthesis.

The immune response to helminthic parasites promotes Th2 responses tounrelated antigens. Infestation with helminthic parasites, which allinduce Th2-type inflammation, can modulate the Th1 immune response tounrelated concomitant parasitic, bacterial and viral infections(Kullberg et al., 1992, J. Immunol. 148:3264). Patients infected with S.mansoni mount more of a Th2-like response to tetanus toxoid immunizationthan the usual Th1 or Th0 (Sabin et al., 1996, J. Infec. Dis. 173: 269).Ethiopian immigrants with a high prevalence of helminthic infectionshave eosinophilia and a propensity to respond to PHA with Th2, ratherthan Th1 cytokines (Bentwich et al., 1996, Clin. Exp. Immunol. 103:239).

Animal experimentation supports this contention. Mice infected withMycobacterium avium develop chronic Th1-type granulomatous inflammationin the lungs and liver. Splenocytes and granuloma cells from theseinfected animals normally produce IgG2a and IFN-γ, and no IL-4 or IL-5.However, mice infected with S. mansoni after the establishment ofMycobacterium avium infection form mycobacterial granulomas containingeosinophils. Also, splenocytes and granuloma cells from co-infected micesecrete more IgG1 and much less IgG2a. The cytokines released from thesecells both constitutively or after mycobacterial antigen stimulationinclude IL-4 and IL-5, and much less than normal quantities of IFN-γ.

There are other examples. Infection of mice with S. mansoni delaysclearance of vaccinia virus and alters responsiveness to sperm whalemyoglobin. Mice also develop a Th2 response when infected with themicrofilariae, Brugia malayi, or immunized with a soluble filarialextract from this parasite. The ongoing Th2 response to this helminthantigen modulates the Th1 response to mycobacterial antigen. Moreover,Nippostrongylus brasiliensis, a murine intestinal nematode, stimulatesTh2 activity. Nippostrongylus delays kidney graft rejection in rats.

EXAMPLES

The invention is illustrated by the following nonlimiting exampleswherein the following materials and methods are employed. The entiredisclosure of each of the literature references cited hereinafter areincorporated by reference herein.

Example 1 General Methods

Animals: Colonies of 129/SV IL-10−/− mutant mice, and appropriatecontrol animals are maintained are housed in facilities maintained as aspecific pathogen-free environment according to standard methods.

Parasite Maintenance, Animal Infection, Production of Schistosome Eggs:The maintenance of T. muris and the method used for infection are asdescribed by Else and Wakelin, 1990, Parasitology, vol. 100, part 3:479.

Schistosome eggs were harvested from the livers of schistosome-infectedhamsters and stored as described by Elliott, 1996, Methods: A Companionto Methods in Enzymology, 9:255. Five infected hamsters yield about2×10⁶ eggs.

Preparation of T. suis Eggs: the following process was used in thepreparation and harvesting of T. suis eggs. Adult T. suis worms wereisolated from the colon of pigs 7-8 wks after exposure to anexperimental inoculation of T. suis eggs. Embryonated eggs were obtainedby culturing adult worms in vitro, and then the excreted eggs, separatedfrom the culture medium by centrifugation, were placed into 0.2%potassium dichromate solution at 22° C. for 5-6 wks with bubbling toobtain infective first-stage larvae. Eggs were washed twice in sterilewater by centrifugation at 1200×g for 10 min, counted, and re-suspendedin the desired amount of saline based on a calculated dose of 2,500.These eggs were stored for use in the subjects. The ova are stable forat least one year in the refrigerator. To assure infectivity, wemonitored patients for the appearance of ova in the stool aftercolonization. The number of ova in the stool is proportional to theintensity of the infestation. Also, from time to time, we infect pigswith our stored ova to assure continued infectivity.

Infection with M. avium: Mice were infected by injecting 10⁶ colonyforming units (CFU) of Mycobacterium avium (ATCC 25291)intraperitoneally. On day 60 of infection, some mice also received 35 S.mansoni cercariae to induce dual infection.

Schistosome Infection and Isolation of Ova: Some experiments used mice(18-20 g) infected for 8-9 wks with S. mansoni. Mice were infectedsubcutaneously with 40 cercariae from the Puerto Rican strain.

Granuloma Isolation and Dispersal: Granulomas form inschistosome-infected mice because of natural egg deposition, whichbegins at the 6th wk of infection. M. avium also induces granulomas. Westudy liver granulomas isolated from infected mice as described(Elliott, 1996, supra). Granulomas were dispersed to produce single-cellsuspensions. Isolated granulomas were agitated for 35 min at 37° C. in ashaker water bath in RPMI medium containing 5 mg/ml collagenase. Theresidual granulomas were sucked and expelled through a 1 ml syringe toinduce further dissociation. The resulting cell suspension was filteredthrough gauze and washed three times. Cell viability was determined byEosin-Y exclusion. This protocol resulted in a high yield of viableinflammatory cells that show preserved surface molecule expression.

Spleen and MLN Dispersal: Splenocytes and MLN were dispersed by teasingand washing the tissue through a stainless steel mesh. The contaminatingRBC were lysed with H20, and the cells were washed ×3 in RPMI beforeuse.

Induction of TNBS Colitis: Colitis was induced by rectal instillation oftrinitrobenesulfonic acid (TNBS) as described by Neurath et al., 1995,J. Exp. Med., 182: 1281. Briefly, control or parasite-exposed BALB/cmice were fasted for 30 hrs then anesthetized with methoxyflurane. A 1.2mm catheter was advanced 4 cm into the colon and 0.1 ml of TNBS solution(5 mg/ml TNBS (Sigma) in 50% ethanol) was instilled. The animal was heldby the tail for 3 minutes to insure uniform contact with colonic mucosa.

Evaluation of Mucosal Inflammation: To grade intestinal inflammation,tissue was removed at the indicated time points, Swiss-rolled andembedded in paraffin according to standard methods. The sections werestained with hematoxylin and eosin. The degree of colonic inflammationwas graded semiquantitatively from 0 to 4 in a blinded fashion by asingle pathologist using our usual standardize technique (1 9). 0=noinflammation, 1=low level inflammation, 2=intermediate level, 3=highlevel inflammation with wall thickening, 4=transmural infiltration, lossof goblet cells, wall thickening.

ELISAs for Murine Cytokines: ELISAs were performed as described insection VI, above.

Example 2 Th2 Response to S. mansoni Down-Modulates an Ongoing Th1Response to an Unrelated Bacterial Th1-Inducing Antigen

It is well established that Th cell immune responses can polarize intoTh1 or Th2 patterns. This polarization occurs because IFNγ from Th1cells inhibits proliferation of Th2 cells, while IL-4 and IL-10 from Th2cells inhibits development of Th I cells. The following experimentsdemonstrated that schistosomiasis alters the murine Th1 response to anestablished mycobacterial infection.

Mice were co-infected with M. avium and S. mansoni to evaluate the hostresponse to these distinctly different Th1 and Th2 inflammatory stimuli.BALB/cAnN mice develop chronic M. avium infection when injected withthis organism (10⁶ CFU). Sixty days after establishment of themycobacterial infection, the mice were infected with S. mansoni (40cercariae). The mice were killed sixty days later. Control groupsincluded mice receiving either M. avium only for 120 days or S. mansonionly for 60 days. Dispersed splenocytes or isolated granuloma cells fromthese animals were cultured in vitro (4×10⁵ cells/well) for 48 h in thepresence or absence of schistosome egg antigen (SEA, a strong Th2antigen) or mycobacterial antigens purified protein derivative (PPD, astrong Th1-inducing antigen) used at optimal concentration. After theincubation, supernatants were assayed for cytokine or immunoglobulinproduction using ELISAS. The data in FIGS. 1-3 are mean values +/−SD ofthree separate experiments.

Splenocytes from mice infected only with M. avium secreted large amountsof IFNγ following stimulation with PPD (Th1 antigen). Spleen cells fromuninfected control mice produced none. Most importantly, no IFNγ wasdetected in spleen cell cultures from concurrently infected mice (M.avium alone vs. concurrent infection, P<0.001, FIG. 1). Solubleschistosome egg antigen (SEA, Th2 antigen) stimulated only IL-4 and IL-5release from splenocytes of S. mansoni-infected animals. Mice singularlyinfected with M. avium produced no IL-4 or IL-5 in response to PPD orSEA. However, splenocytes from co-infected animals secreted some IL-4following PPD stimulation (FIG. 1).

Granulomas were isolated from the livers of mice infected with M. aviumor S. mansoni, or from animals that had concurrent infection.Concurrently infected animals develop liver granulomas that contain bothschistosome eggs and mycobacteria readily evident on histologicalexamination. Dispersed granuloma cells from these animals were culturedin vitro for 48 h in the presence or absence of SEA or PPD used atoptimal concentration. Granuloma cells from mice only infected with M.avium secreted large amounts of IFNγ following stimulation with PPD. NoIFNγ was detected in granuloma cell cultures from concurrently infectedmice (M. avium alone vs other, P<0.001) (FIG. 2). SEA stimulated IL-4release from granuloma cells of S. mansoni-infected animals. SEA did notpromote IFNγ secretion under any circumstance. Mice singularly infectedwith M. avium produced no IL-4 in response to PPD. However, granulomacells from co-infected animals secreted some IL-4 following PPDstimulation (FIG. 2).

Th1 responses promote IgG2a production, whereas Th2 reactions enhanceIgG1 and IgE. FIG. 3 shows that mice infected with M. avium have highserum IgG2a levels. Yet, co-infected animals have normal serum IgG2aconcentrations, but increased IgG1 and IgE levels. These data takentogether show that a Th2 response to a helminthic infection candown-modulate the ongoing host response to even a strong Th1-inducingorganism like M. avium.

Example 3 Colonization with Intestinal Helminths or Exposure to theirOva Attenuates Th1-Type Gut Inflammation in Murine TNBS-Induced Colitis

Rectal instillation of TNBS in 50% ethanol induces a colitis in micethat shares features with Crohn's disease. The colonic inflammation ischaracterized by infiltrating CD4+ T cells with elevated IFNγ mRNAexpression. Lamina propria T cells from TNBS treated mice secrete 50fold more IFNγ and 5 fold less IL4 than T cells from controls (Neurathet al., 1995, supra). Lamina propria mononuclear cells secrete 30 foldmore TNFα than cells from control mice (Neurath et al., 1997, Eur. J.Immunol., 27: 1743). Importantly, TNBS colitis can be prevented orimproved by treatment with anti-IL-12 (Neurath et al., 1995, supra),anti-TNFα (Neurath et al., 1997, supra), or rIL-10 (Duchmann et al.,1996, Eur. J. Immunol., 26: 934). TNBS-induced colitis also can beprevented by previous oral exposure to the hapten (Elson et al., 1996,J. Immunol., 157: 2174) probably by increasing mucosal IL-4, IL-10 andTGFβ responses (Neurath et al., 1996, J. Exp. Med., 183: 2605).

We established this TNBS colitis model using BALB/c mice in ourlaboratory. Rectal administration of TNBS (0.1 ml of 5 mg/ml stock) in50% ethanol reproducibly produced colitis in these animals. For each ofthe TNBS experiments discussed below, parasite-exposed and controlanimals were administered the same preparation of TNBS on the same dayby the same operator who was blinded to the treatment group.

A. Schistosomiasis Inhibits IFNγ Release from MLN and Spleen Cells ofTNBS Treated Mice.

As described above, schistosomiasis inhibits the murine Th1 response toan established mycobacterial infection. We extended these observationsto determine if schistosome infection alters the Th1 response in TNBStreated mice. Mice were infected with 35 S. mansoni cercariae by sc.injection. Worms mature and begin to lay eggs about 6 weeks afterinitiation of infection. Two weeks later (at 8 wks of infection), micewere treated with TNBS. The capacity of MLN and spleen cells to makeIFNγ in response to T cell stimulation (anti-CD3) was examined severaldays later. As shown in Table 1, natural schistosome infection stronglyinhibits IFNγ release from mesenteric lymph node (MLN) and spleen cellsof TNBS-treated mice. TABLE 1 Schistosomiasis Inhibits IFNγ Productionfrom MLN and Splenic T Cells of TNBS-Treated Mice α-CD3 stimulated IFNγ(ng/ml) Group MLN cells Spleen cells Uninfected mice given TNBS 3.2 ±0.7 44.1 ± 2.3 Schistosome-infected mice given TNBS   1.9 ± 0.3^(a)  4.2 ± 0.4^(b)Mean IFNγ (ng/ml) ± SE of triplicate determinations measured by ELISA.^(a))p < 0.1^(b))p < 0.05Cultures contained 10⁶ dispersed MLN or Spleen cells/well incubated in200 μl medium for 48 hrs at 37° C. in the presence of anti-CD3 (1 μg/ml)(2C11)Results are representative of two experiments.B. Exposure to Schistosome Ova Inhibits IFNγ Release from MLN and SpleenCells of TNBS Treated Mice.

In schistosomiasis, it is exposure to the parasite ova rather than adultworms that induces a Th2 response. Schistosome infection does not inducestrong Th2 responses until the worms mature and begin to lay eggs(Grzych et al., 1991, J. Immunol., 146: 1322). Mice exposed to intactschistosome eggs in the absence of natural infection develop a strongTh2 response (Oswald et al., 1994, J. Immunol., 153: 1707). Theseobservations suggest that exposure to schistosome eggs in the absence ofnatural infection may induce Th2 and suppress Th1 responses.

We tested the hypothesis that pre-exposure to schistosome eggs wouldinhibit Th1 responses without requiring infection by adult worms. Micewere inoculated twice with 104 schistosome eggs by intraperitoneal (ip)injection at 14 and 4 days prior to rectal challenge with TNBS. Thesetimes were chosen to model the continuous egg deposition that occurs innatural infection. Mice not exposed to parasite eggs but treated withTNBS served as controls. The eggs were previously frozen and were notviable at the time of injection. Once again, the capacity of MLN andspleen cells to make IFNγ in response to T cell stimulation (anti-CD3)was examined several days after TNBS instillation. Like naturalschistosome infection (Table 1), ip. egg exposure inhibited IFNγproduction from MLN and splenic T cells of TNBS-treated mice as shown inTable 2. TABLE 2 Exposure to Schistosome Eggs Inhibits IFNγ Productionfrom MLN and Splenic T Cells of TNBS-Treated Mice α-CD3 stimulated IFNγ(ng/ml) Treatment MLN cells Spleen cells TNBS alone 4.65 ± 0.02 47.1 ±3.0 Eggs ip. and TNBS   2.19 ± 0.11^(a)  21.8 ± 3.4^(b)Mean IFNγ (ng/ml) ± SD of triplicate determinations measured by ELISA.^(a))p < 0.5.Cultures contained 10⁶ dispersed MLN or spleen cells/well incubated in200 μl medium for 48 hrs at 37° C. in the presence of anti-CD3 (1 μg/ml)(2C11).Results are representative of three separate experiments.C. Exposure to Schistosome Ova Protects Mice from TNBS-Induced Colitis.

Inhibiting development of the mucosal Th1 response can attenuateTNBS-induced colitis. Prior exposure to schistosome eggs inhibits Th1cytokine secretion by MLN and splenic T cells. We next ascertained if ipinjection of schistosome eggs would inhibit TNBS-induced colitis. Eggswere injected as above followed by TNBS treatment. Egg treatmentdramatically reduced cumulative mortality in three separate experimentsfrom 60% (16/27) in the control group to 22% (6/27) in egg-exposed mice.Intestinal inflammation was scored on a 4-point scale as detailed inGeneral Methods, above. In the mice that survived, egg treatmentattenuated intestinal inflammation from 3.1±0.5 (mean ±SD) in thecontrol group to 1.3±0.3 in the egg exposed mice (p<0.05, FIG. 4).Subsequent experiments showed that the largest difference between groupswas evident at 3 days after TNBS treatment. Other experiments carriedout to 14 days after TNBS instillation showed that egg exposure affordedprolonged protection. These data indicate that schistosome eggs protectmice from developing fatal colitis by inhibiting mucosal Th1 responses.

D. Intestinal Helminths Induce Host Th2 Responses.

It is probable that helminthic parasites other than S. mansoni canmodulate host Th1 responses. Expression of protective immunity tointestinal nematodes is CD4 T cell-dependent. Mice expel worms or limitinfection by mounting a Th2 response. Worm expulsion does not appear toexclusively depend on intestinal eosinophilia nor mucosal mastocytosis.IL-4 may have a critical role in worm expulsion, since treatment withblocking anti-IL-4 or anti-IL-4 receptor mAb promotes worm retention(Else et al., 1994, J. Exp. Med., 179: 347). Conversely, treatment withIL-4 promotes worm clearance (FIG. 4).

T. muris lives in the colon of the murine host. It is related toTrichuris trichiura, a parasite carried by nearly one billion peopleduring their lives (Grencis et al., 1996, Gastroenterology Clinics ofNorth America, 25: 579). Ingestion of eggs initiates infection. The eggsrelease larvae that penetrate the cecal epithelium. They then matureinto adult worms. The parasite does not replicate within the hostallowing us to control the intensity of infection (Bancroft et al.,1994, Eur. J. Immunol., 24: 3113).

We used T. muris to down-modulate intestinal Th1 responsiveness. T.muris infection is established by oral gavage with 250 embryonated eggscontaining viable larvae. BALB/c mice can harbor T. muris and naturallyexpell the worms within 4 wks of infection. We treated T. muris- orsham-infected mice with rectal instillation of TNBS 4 wks afterinitiation of infection. Prior colonization with T. muris reducedcumulative mortality in two separate experiments from 58% (7/12) in thesham infected group to 21% (3/14) in the parasite exposed group.Furthermore, mice previously colonized with T. muris develop attenuatedTNBS colitis (0.92±0.5, mean ±SD) as compared to sham-infected mice(3.13±0.63, p<0.05). These data indicate that prior exposure tointestinal parasites (T. muris) protect mice from developing severeTh1-mediated colitis.

Example 4 IL-10 Gene Disruption does not Significantly Alter theHost/Parasite Interaction

IL-10 is an important immunoregulatory cytokine that down modulatesmacrophage activation and accessory cell function (Moore et al., 1993,Ann. Rev. Immunol., 11: 165). Mice rendered IL-10 deficient by targetedgene disruption (IL-10−/−) develop a chronic enterocolitis that isinfluenced by colonic flora (Kuhn et al., 1993, Cell, 75: 263). Theintestinal inflammation is attenuated by treatment with anti-IFNγantibody demonstrating that the colitis results from overly exuberantTh1 responses to colonic contents (Berg, et al., 1996, J. Clin. Invest.,98: 1010). These mice serve as excellent models for spontaneous colitissimilar to that of Crohn's disease. In this example we have usedIL-10−/− mice on the 129 and C57B1/6 backgrounds.

A. Disruption of the IL-10 Gene does not Alter Host Susceptibility toParasite Colonization.

IL-10−/− mice spontaneously develop chronic intestinal inflammation. Apotential concern was that this inflammation might prevent effectivecolonization with T. muris. We have found, however, that we routinely(>95%) obtain patent colonization with T. muris in IL-10−/− mice on boththe 129 and C57B1/6 backgrounds. To date, we have colonized more than 40IL-10−/− mice with T. muris. IL-10 deficient mice also can harbor S.mansoni that mature to adult worms and lay eggs (Wynn et al., 1998, J.Immunol., 160: 4473). We have confirmed these findings (see below).

B. IL-10−/−Mice can Mount Th2 Responses to Parasites.

Mice infected with the intestinal nematode N. brasiliensis developTh2-type inflammation to the parasite with production of IL-4, IL-5 andIL-10. N. brasiliensis can colonize IL-10−/− mice and stimulate anappropriate intestinal Th2 response (Kuhn et al., 1993, supra). Weinfected IL-10−/− mice with S. mansoni to examine whether they mount aTH2 response.

Table 3 shows that splenocytes from IL-10 gene-disrupted mice colonizedfor 8 wk with S. mansoni secrete large amounts of IL-4 upon stimulationwith schistosome egg antigen (SEA) or anti-CD3. The granulomas thatsurround schistosome eggs in these mice contain the usual highpercentage (45-50%) of eosinophils, and make IL-4 and IL-5. Thus, theyshow an effective Th2 response. These data indicate that exposure tohelminthic parasites like S. mansoni will induce a strong Th2 responseeven in the absence of IL-10. TABLE 3 Schistosomiasis Induces a Th2Response in IL10 Deficient Mice Spleen Cells from a Schistosome-infectedMice SEA- α-CD3 Cytokine Unstimulated Stimulated Stimulated IL-4 (ng/ml)Wild Type 0.07 ± 0.08 0.21 ± 0.02 3.94 ± 0.12 IL-10—/— 0.41 ± 0.10 2.56± 0.23 5.06 ± 0.33Mean IL-4 (ng/ml) ± SD of triplicate determinations as measured byELISA.Cultures contained 10⁶ cells/well incubated for 48 hrs at 37° C. in 200μl medium in the absence or presence of schistosome egg antigen (SEA 5μg/ml) or anti-CD3 (1 μg/ml) (2C11).Results are representative of two separate experiments using a minimumof four wild type and KO mice in each experiment.D. Helminthic Th2-Conditioning Inhibits Natural Development of MucosalInflammation in IL-10−/− Mice.

We showed that IL-10 deficient mice could harbor helminthic parasitesand mount a strong Th2 response. Because IL-10−/− mice can develop Th2responses and harbor intestinal parasites, they serve as excellentmodels to study the effect of parasite exposure on spontaneous orongoing colitis. IL-10 is an important anti-inflammatory cytokine. It ispossible that disruption of this essential immunoregulatory circuit willprevent mucosal Th2 conditioning by parasites. Present evidenceindicates that infection with T. muris impedes the spontaneous colitisthat develops in IL-10−/− mice. Animals (6-wk-old) received T. muris orsham infection and were killed 6 wks later. Colonic inflammation of T.muris or sham-infected IL-10−/− mice was scored on a 4-point scale asdetailed in General Methods, above. Prior infection with T. murisattenuated intestinal inflammation from 3.0±0.3 (mean ±SE) in thesham-infected group to 2.2±0.1 (p<0.05) in the parasite exposed IL-10−/−mice. These data demonstrate that prior exposure to helminthic parasitesattenuates spontaneous colitis in IL-10 deficient mice.

Example 6 Intestinal Colonization with Trichuris suis Down-ModulatesDisease Activity in Patients with Crohn's Disease

Our major hypothesis is that failure to obtain helminthic infections andexperience mucosal Th2 conditioning during childhood predisposes toCrohn's disease and other autoimmune diseases which are the result ofoverly active Th1 reactions. An important corollary hypothesis is thatcolonization with intestinal parasites will down modulate establishedinflammation. We expect that this is possible because concurrentschistosomiasis impedes Th1 responses in an established M. aviuminfection (see above). We tested this hypothesis by colonizing patientswith Crohn's disease with Trichuris suis and assessing improvement indisease activity. T. suis, the porcine whipworm, is closely related toT. trichiura, a human intestinal helminth common in underdevelopedcountries. The whipworm is a potential agent for therapy. The naturalhuman parasite Trichuris trichiura is a very small organism that residesin the colon by attachment to the mucosa. Ordinary colonization usuallyproduces no symptoms and causes no health problems for the host. This isthe case in millions of colonized people throughout the world, but in aminority, heavy infestation produces diarrhea, bleeding and irondeficiency anemia. It is of interest that the parasite's life cycle issuch that the host is not self-infected. The eggs would require a soilphase for maturation to become infective and then must be re-ingested toincrease the parasitic load for an individual. Thus, T. trichiurainfestation will not increase within the host unless eggs in the soilare ingested. The agent is readily and effectively treated with threedays of mebendazole. The human whipworm could be used to colonize thehost and be considered as an experimental agent to modify the immuneprocess in Crohn's disease.

However, Trichuris trichiura poses a potential health problem for thehost. Because the human is the only natural host, eggs for experimentaluse would have to be harvested from other humans, creating the potentialfor transmission of other human infectious diseases to the experimentalsubject. Therefore, we instead used a closely related animal parasite,the porcine whipworm (Trichuris suis), that has the potential totemporarily colonize a human host without causing symptoms, disease, aco-infectious disease or a public health hazard.

The porcine whipworm is closely related to the species infecting humans.The two organisms are of the same family and are morphologicallysimilar, but they belong to a different species and can be distinguishedmorphologically, developmentally and clinically. The porcine whipwormovum is slightly larger, has a different shaped spine, and the rate ofdevelopment from an egg to an adult is slower than that of Trichuristrichiura (Beer, 1976, Res. Vet. Sci., 20:47). Importantly, we canobtain infective parasite ova from SPF animals.

We produced a supply of infective T. suis eggs as described above. Theeggs were tested to confirm absence of contamination with entericpathogens (e.g. Shigella, Salmonella, Campylobacter, Yersinia, andenterotoxic E. coli) and viruses (e.g. CMV, HSV, VZV, adenovirus andenteroviruses).

Two patients with advanced, medication-resistant Crohn's disease werecolonized with T. suis. They tolerated the parasite with little or nosymptoms attributable to this organism. Table 4 shows that afterachieving patent colonization, both participants had a drop in CDAIvalues, diarrhea, and inflammatory indices. This result indicates thathelminths are useful for modulating abnormal immune responses,including, but not limited to Crohn's disease. TABLE 4 Patients withCrohn's disease may benefit from colonization with T. Suis. PatientParameter Baseline^(a) Week 2^(b) Week 4^(b) 1 CDAI^(c) 250.2 269.9147.4 Stools/week 67 101 49 ESR^(d) 17 11 4 C-reactive <0.5 <0.5 <0.5protein 2 CDAI 341.3 169.6 136.7 Stools/week 77 27 25 ESR 19 7 NDC-reactive 0.9 <0.5 <0.5 protein^(a))Stable values at entry into study.^(b))Values at 2 and 4 weeks after initiation of colonization with T.suis.^(c))Crohn's disease activity index.^(d))Erythrocyte sedimentation rate.ND; not determined.

OTHER EMBODIMENTS

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing detailed description is providedfor clarity only and is merely exemplary. The spirit

1-10. (canceled)
 11. A method of treating inflammatory bowel disease(IBD) in an individual comprising, identifying an individual with IBD inneed of treatment; administering to the individual a compositioncomprising a T. suis helminthic parasite preparation in an amountsufficient to treat said IBD.
 12. The method of claim 11, furthercomprising the step of measuring the Th1 immune response in saidindividual following administration of said T. suis helminthic parasitepreparation.
 13. The method of claim 11, further comprising the step ofmeasuring a decrease in a clinical parameter of IBD.
 14. The method ofclaim 11 wherein the T. suis helminthic parasite preparation comprises aparasite selected from the group consisting of parasite, parasiteextract, parasite eggs, parasite egg extract, parasite larvae, andparasite larvae extract.
 15. A method of treating IBD in an individualcomprising, identifying an individual with IBD in need of treatment;administering to the individual a composition comprising a T. suishelminthic parasite preparation in an amount sufficient to treat saidIBD; and monitoring the treatment of IBD in said individual by measuringa decrease in a clinical parameter of IBD.
 16. The method of claim 15,further comprising the step of measuring the Th1 immune response in saidindividual following administration of said T. suis helminthic parasitepreparation.
 17. A method of treating IBD in an individual comprising,identifying an individual with IBD in need of treatment; administeringto the individual a composition comprising a T. suis helminthic parasitepreparation in an amount sufficient to treat said IBD; and monitoringthe treatment of IBD in said individual by measuring a decrease a Th1immune response.
 18. The method of claim 15, further comprising the stepof measuring a decrease in a clinical parameter of IBD in saidindividual following administration of said T. suis helminthic parasitepreparation.
 19. A method of treating IBD in an individual comprising,identifying an individual with IBD in need of treatment; administeringto the individual a composition comprising a helminthic parasitepreparation comprising a helminthic parasite selected from the groupconsisting of T. suis, S. mansoni, H. polygyrus, T. spiralis, and N.americanus in an amount sufficient to treat said IBD.
 20. The method ofclaim 19, further comprising the step of measuring the Th1 immuneresponse in said individual following administration of said helminthicparasite preparation.
 21. The method of claim 19, further comprising thestep of measuring a decrease in a clinical parameter of IBD.
 22. Themethod of claim 19 wherein the helminthic parasite preparation comprisesa parasite selected from the group consisting of parasite, parasiteextract, parasite eggs, parasite egg extract, parasite larvae, parasitelarvae extract, parasite cercariae and parasite cercariae extract.